Background: Rotator cuff tears (RCTs) induce chronic muscle weakness and shoulder pain. Treatment of RCT using surgery or drugs causes lipid infiltration and fibrosis, which hampers tissue regeneration and complete recovery. The pluripotent stem cell-derived multipotent mesenchymal stem cells (M-MSCs) represent potential candidate next-generation therapies for RCT. Methods: The difference between M-MSCs and adult-MSCs was compared and analyzed using next-generation sequencing (NGS). In addition, using a rat model of RCT, the muscle recovery ability of M-MSCs and adult-MSCs was evaluated by conducting a histological analysis and monitoring the cytokine expression level. Results: Using NGS, it was confirmed that M-MSC was suitable for transplantation because of its excellent ability to regulate inflammation that promotes tissue repair and reduced apoptosis and rejection during transplantation. In addition, while M-MSCs persisted for up to 8 weeks in vivo, they significantly reduced inflammation and adipogenesis-related cytokine levels in rat muscle. Significant differences were also confirmed in histopathological remission. Conclusions: M-MSCs remain in the body longer to modulate immune responses in RCTs and have a greater potential to improve muscle recovery by alleviating acute inflammatory responses. This indicates that M-MSCs could be used in potential next-generation RCT therapies. Rotator cuff tears (RCTs) are commonly associated with fibrosis, muscle atrophy, and inter- and intra-muscular fat infiltration, which are commonly referred to as “fatty degeneration” . They arStem cell-based therapy can be used to treat fatty degeneration and inflammation. Mesenchymal stem cells (MSCs) are a population of multipotent adult stem cells found in multiple compartments of the body. It has been reported that MSC has an inhibitory effect on the proliferation, differentiation and activation of immune cells and can be usefully applied to the treatment of inflammation-related diseases . MSCs caIn this study, we hypothesized that M-MSCs would be good candidates for stem cell-based therapies, and that they would affect inflammation relief and muscle recovery in the RCT model. Therefore, we evaluated the transplant suitability of adult-MSCs and M-MSCs using a principal component analysis (PCA) and gene ontology (GO) analysis, and evaluated their ability to suppress immune rejection through mixed lymphocyte response. In addition, we investigated the potential of M-MSCs as a next-generation stem cell-based therapeutic agent in a rotator cuff tear model of M-MSCs by conducting an histological and immunohistochemical analysis and gene expression analysis by creating an RCT model.2.The M-MSCs were obtained from embryoid bodies (EBs) of hPSCs as previously described ,13. BrieFor EB formation, the hPSC colonies were harvested using dispase treatment  and were grown in suspension culture for 2 days with the same hPSC culture medium except for bFGF. To isolate MSC-like cells, porous membrane transwell inserts with 8 μm pores were used. The upper compartment of the inserts was coated with 0.1% gelatin, and the EBs were attached in EBMTM-2 Basal Medium supplemented with EGMTM-2 MV Microvascular Endothelial Cell Growth Medium Supplements SingleQuotsTM  (EGM2 MV media) for 5 days. The cells that migrated to the lower compartment of the inserts formed colonies, which were gently scraped and subcultured onto a new 100 mm dish in the same EGM2 MV media, and were called M-MSCs.The M-MSCs were cultured in EGM2 MV media and were expanded for fewer than 10 passages to ensure that multipotency was preserved. The medium was changed every two days. Each cell was harvested when it reached 95% confluency.® Ribospin™  according to the manufacturer’s protocol and all RNA samples were then determined as high and comparable quality. Libraries were generated according to standard procedures using a TruSeq Stranded mRNA LT Sample Prep Kit  according to a TruSeq Stranded mRNA Sample Preparation Guide, Part #15031047 Rev. E (Illumina Inc.). The libraries were sequenced on a NovaSeq 6000 S4 (Illumina Inc.) according to a NovaSeq 6000 System User Guide Document #1000000019358 v02 (Illumina Inc.) in a sequencer NovaSeq (Illumina Inc.). The set of RNA-seq data for M-MSCs was derived from five biological samples.RNA-sequencing (R-seq) was conducted to compare M-MSCs to bone marrow-derived mesenchymal stem cells (BM-MSCs) and adipose-derived mesenchymal stem cells (AD-MSCs). Firstly, total RNA was extracted from the M-MSCs using GeneAllhttps://bedtools.readthedocs.io/en/latest/ accessed on 7 February 2022). Read mapping and expression quantification were performed separately for each sample.The trimmed reads scoring values of over 98% of the average Q20 and over 95% of the average Q30 in Phred quality score using BBDuk (part of BBtools) were kept. The remaining reads were mapped to the reference genome sequence  hg19, annotation RefSeq_2017_06_12) using Bowtie2 . CalculaAdditionally, from National Center for Biotechnology Information Gene Expression Omnibus (NCBI GEO), transcriptomic data of BM-MSCs  and AD-MSCs  were obtained. The gene expression levels then were quantile normalized using R 4.0.3 .r) and significance levels were calculated using the function rcorr and the function chart. Correlation found in the R statistical programming language and the correlogram was plotted using the R packages. The strength of associations were categorized as follows: 0.90 to 1.00, very highly correlated; 0.70 to 0.90, highly correlated; 0.50 to 0.70, moderately correlated; 0.30 to 0.50, lowly correlated; and 0.00 to 0.30, negligibly correlated [Pearson’s product–moment correlation coefficient rho  was set to 0.4.Upregulated differentially expressed genes (DEGs) in the M-MSCs were selected based on fold changes (FCs) 4 times higher than BM-MSCs or AD-MSCs while downregulated DEGs based on FCs were 0.25 times lower. For the selected DEGs, a Gene Ontology (GO) network and Enrichment Pathway analysis were performed using ClueGO 2.5.7 and CluePedia 1.5.5 on Cytoscape 3.7.2  on Java script 1.8.0_162  [https://wikipathways.org accessed on 10 February 2022) [https://sourceforge.net/projects/mev-tm4/files/mev-tm4 accessed on/ 21 May 2021).Additional analyses for specific GOs were conducted with the GO term GO: 0043069 ‘negative regulation of programmed cell death’ (EMBL-EBI) and GO term WP2328 ‘Allograft Rejection (Homo sapiens)’ (ry 2022) . The gen7 cells/mL) were treated with 10 μg/mL mitomycin C at 37 °C for 1 h, followed by 5 extensive washes with FBS-containing RPMI 1640 medium. Responder splenocytes from BALB/c mice and stimulator splenocytes from different strains of mice were resuspended in RPMI 1640 containing 10% FBS, 2 mM glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin, 1 mM sodium pyruvate, 20 mM HEPES, and 50 uM 2-mercaptoethanol. Each responder and stimulator cell population were seeded in triplicate at a concentration of 105 cells/100 μL/well, in 96-well. MSCs were also added to the MLR at the same number to obtain a 300-μL final volume. In mitogen proliferative assays, responder splenocytes were incubated with 5 μg/mL concanavalin A. After 5 days, the proliferation of responder cells was measured using a CCK assay.Splenocytes were isolated from mouse spleen by disaggregation into 10 mL RPMI 1640 medium. Erythrocytes were lysed with RBC lysis buffer  and subsequently washed 3 times in RPMI 1640. Stimulator splenocytes  rats were purchased from ORIENT Bio Inc. . These rats were acclimated for a week and used at the age of 8-weeks-old. During the experiment, all rats were provided with tap water and pellet  ad libitum. All animals were maintained at a temperature of 22 ± 2 °C with a humidity of 55 ± 5% and a light/dark cycle of 12 h/12 h.6 cells per animal. The normal group refers to no surgery or cell treatment, and the control group refers to the hDF-treated group as a control for cell treatment. The skin was closed with 6-0 nylon suture, without repairing the tendon. The rats were allowed unrestricted cage activity. At 24 h, 72 h, 7 days, 4 weeks, and 8 weeks after surgery, each rat was euthanized with CO2 gas inhalation.The RCT model was induced according to the previously described method  with som6 cells, and 200 μL was prepared and injected at the time of surgery. After sampling at 24 h, 72 h, 7 days, 4 weeks, and 8 weeks, paraffin sections were performed, and the labeled cells were monitored using a microscope .M-MSCs, BM-MSCs and hDF cells were adhered to and cultured in a 100 mm dish, and when the density reached 90%, the media was removed and washed with DPBS. Cell Tracker™ CM-DiI dye  stock was prepared as recommended by the manufacturer. The stock was diluted to 1 μM in DPSB and placed in a cell dish. Incubation was performed at 37 °C for 15 min and then at 4 °C for 15 min. Then, the staining solution was removed, washed twice with DPBS, and the cells were detached using trypsin. The cell suspension was centrifuged, adjusted to 3 × 10A grip strength meter  was used to assess the forelimb grip strength of the rats. Rats were lifted by the tail and induced to grasp a mesh attached to a digital force gauge with its right forelimb. The mesh was 3 × 4.75 square inches with 1-square inch grids. The bars of the grids were 1/8-inch thick. The mesh was oriented horizontally. The animal was gently lifted over the mesh by holding its tail and then lowered toward the mesh while its tail, body, and forelimb were kept in a line and perpendicular to the mesh when the animal reached out to hold onto the mesh. The tension reading of the digital force gauge was defined as the grip strength before the rat released the net. Three consecutive tests were performed on each rat and the mean maximum limb muscle strength value  was obtained. It was measured at 24 h, 72 h, 7 days, 4 weeks, and 8 weeks after surgery, and the value that reached the normal measured value was defined as a percentage.® Ribospin™ . First-strand cDNA was then synthesized using the extracted total RNA as a template with AccuPower® RT PreMix  according to the manufacturer’s protocols. The resulting cDNA was subjected to qPCR using AccuPower® 2× GreenStar™ qPCR Master Mix . Primer sequences and detailed experimental conditions for the amplifications of GAPDH, IL-1b, IL-6, TNF-a, Cox2, Ngf, Ptges, Ppar-r, MyoD, Myf5 and cebpa are shown in target gene − CtGAPDH). Relative fold changes of target gene expressions were determined using the comparative 2−ΔΔCt method (ΔΔCt = ΔCtM-MSC − ΔCtcontrol) [Total RNA was extracted from the supraspinatus and infraspinatus muscles using GeneAllcontrol) .Whole muscular tissues from the RCT models treated with M-MSCs, BM-MSCs or hDFs were excised at 72 h after surgery for comparison with a normal rat. For the semi-quantitative protein antibody array, proteins (1300 to 1800 μg) were extracted from the muscular tissues. A Rat L2 Antibody Array slide  was dried for 2 h at room temperature and was incubated with 400 μL of blocking solution at room temperature for 30 min. After decanting the blocking buffer from each sub array, 400 μL of the diluted samples was added and samples were incubated for 2 h at room temperature. After decanting the samples, each array was washed three times with 800 μL of 1× wash buffer I at room temperature for 5 min with shaking. The glass chip assembly was placed into the container and a sufficient amount of 1× wash buffer I was added to submerge the entire glass chip for 10 min with shaking twice. The advanced washing step with 1× wash buffer II was repeated. 1× biotin-Conjugated Anti-Cytokine antibodies were prepared and incubated for 2 h at room temperature with gentle shaking, and washed with 150 μL of 1× wash buffer I at room temperature with shaking. A 1× Cy3-Conjugated Streptavidin stock solution was added and incubated for 2 hr at room temperature with gentle shaking, and was washed with 1× wash buffer I for 10 min at room temperature twice. After washing, the slide was rinsed with deionized water using a plastic wash bottle and was centrifuged at 1000 rpm for 3 min to remove water.The slide scanning was performed using a GenePix 4100A Scanner . The slides were completely dried before the scanning and scanned within 24-48 h. The slides were scanned at a 10 μm resolution, with optimal laser power, and PMT. After obtaining the scanned image, they were gridded and quantified with GenePix Software . After analysis, the data about protein information were annotated using UniProt DB and were quantile normalized.To evaluate histopathological changes, the supraspinatus and infraspinatus muscles of each rat were freshly excised and fixed with 10% neutral buffered formalin for 24 h. Tissues were processed using routine tissue techniques and embedded in paraffin in cross-sections for the supraspinatus and longitudinal sections for the infraspinatus. Paraffin-embedded specimens were sliced into 5-μm-thick sections. Sections were then transferred to adhesive microscope slides . Deparaffinized muscle sections were stained with hematoxylin and eosin (H and E), Masson’s trichrome (MT) and Toluidine blue (TB). Additionally, immunohistochemical staining (IHC) was conducted using an anti-CD68 antibody . All stained sections were then examined with a light microscope  to assess histological changes including mast cell infiltration, and CD68-positive macrophage infiltration. Three sections per animal were used for histological examinations.The transcriptomic data were analyzed to find their Pearson product–moment correlation coefficient rho (r) and their significance levels using the functions rcorr and chart.Correlation within R 4.0.3 , and the PCA was conducted using the functions prcomp and autoplot (pam) within R. The values of the correlation and significance level are displayed for those with a P value of less than 0.01.p < 0.05, GO trees between 4 and 6, minimal = 3 genes per node, and Kappa Score = 0.4.After the selection of the upregulated DEGs in the M-MSCs based on FC > 4 and downregulated DEGs based on FC < 0.25, the selected DEGs were analyzed using ClueGO 2.5.7 and CluePedia 1.5.5 on Cytoscape 3.7.2  on Java script 1.8.0_162  with GO_BiologicalProcess-EBI-UniProt-GOA-ACAP-ARAP_08.05.2020_00h00: 17972 , a Additionally, the transcriptomic data were transformed with log2-FCs and Quality Threshold Clustering (QTC) was then applied using the TIGR MultiExperiment Viewer (MeV) 4.9.0  with Pearson correlation matrix, a cluster diameter = 0.5, and minimal = 5 genes per cluster. The data are shown as mean ± standard deviation (SD).p-value of less than 0.05.While the data of qPCR are shown as mean ± standard error of the mean (SEM), the data of the MLR, the grip strength, the areas of fibrosis, the number of mast cells, and the number of CD68+ macrophages are shown as mean ± SD. All statistical analyses were performed using GraphPad Prism . Statistical significance was considered at a All animal experiments were performed in accordance with relevant guidelines and regulations of the Institutional Animal Care and Use Committee of Konkuk University (IACUC authorization no. KU20008) accredited for laboratory animal care by the Ministry of Food and Drug Safety of South Korea.r = 0.42 ± 0.05). However, the correlation between M-MSCs and AD-MSCs was negligible (r = 0.25 ± 0.02).A PCA of several samples of M-MSCs, BM-MSCs, and AD-MSCs was performed to establish the consistency of M-MSCs as cell lines . SamplesThe characteristics of M-MSCs compared with adult MSCs were also confirmed via DEG analysis . As showTo confirm their application as a cell therapy for transplantation, the characteristics of M-MSCs were analyzed for specific GOs compared with adult MSCs . RegardiSimilar to the NGS results, a mixed lymphocyte reaction was performed to determine whether or not M-MSCs had low immunogenicity compared with other adult MSCs. The results showed that all MSCs exhibited limited activity of responder splenocytes caused by stimulator splenocytes. M-MSCs exhibited significantly lower levels of immune rejection than AD-MSCs, and showed immunogenicity comparable to gold standard BM-MSCs C. Based We compared the grip strength for each administered cell and time. All values were expressed as percentages of the values reached to the values of the normal group each time. No differences were found between cells at 24 h and 72 h after surgery. Starting from day 7, the MSC group showed faster muscle recovery than the hDF group. The M-MSCs group showed a more significant difference than the BM-MSCs group B. ComparH and E and MT staining were performed for histological analysis. Fat infiltration, histological atrophy, and fibrosis were confirmed via H and E staining. In the supraspinatus muscle, histological atrophy was observed for up to 7 days in the M-MSCs group, whereas no fibrosis or fat infiltration was detected. In the BM-MSCs group, some fat infiltration was observed at 4 weeks, but overall it was less apparent. However, histologic atrophy and muscle fibrosis were observed until week 4. The histological atrophy was not fully reversed at week 8. The hDF group showed serious fat infiltration from day 7, and the fibrosis and muscle atrophy progressed from 72 h until week 8 A1.The M-MSC group showed atrophy of infraspinatus until week 4, although fibrosis or fat infiltration was confirmed at 7 days, suggesting that the M-MSCs group recovered adequately to approach the normal group from week 4. In the case of the BM-MSCs group, the fibrosis or muscle atrophy recovered after 7 days, similar to that of the M-MSCs group. However, the fat infiltration was severe from day 7 until week 4. In the case of the hDFs group, muscle atrophy, fibrotic muscle, and fat infiltration were observed at up to 8 weeks A2.Fibrosis was confirmed by MT staining. In the supraspinatus muscle, the M-MSCs and BM-MSCs groups showed a significant difference compared with the normal group, accounting for about 20% of the total area of fibrosis until 72 h. The fibrotic area was reduced and recovered to almost the same value. In contrast, in the hDFs group, a significant range of fibrosis was confirmed at up to 8 weeks B1,B2.In the infraspinatus muscle, the M-MSCs group was similar to the supraspinatus muscle, and fibrosis was confirmed for up to 72 h, followed by a decline from day 7 and recovery similar to the normal group after 4 weeks. In the case of BM-MSCs, fibrosis increased significantly compared with the M-MSCs group until day 7, and decreased from week 4. In the case of the hDFs group, fibrosis persisted until week 4, and the maximum fibrosis was confirmed on day 7, followed by a decline similar to the normal group at 8 weeks B3,B4. H Mast-cell infiltration was confirmed via TB staining. In the case of the M-MSCs group, usually less than 5 mast cells per area were found in the supraspinatus, but there was no significant difference compared with the normal group. In the infraspinatus, fewer than 10 mast cells were found at 24 h. A slightly higher number of mast cells than in the normal group was found, but there was little or no significant difference from the normal group after 72 h. In the case of the BM-MSCs group, mast cells were found in the supraspinatus similar to M-MSCs, without a significant difference from the normal group. Inflammation in the infraspinatus was not significantly different compared with the normal group at up to 72 h, and mast cells were confirmed. In the case of hDFs, a significantly higher number of mast cells were confirmed compared with the normal group at up to 7 days, which affected the early stage after surgery A1–A4.Next, macrophage infiltration was confirmed by CD68 staining. In the M-MSCs group, macrophages were found with a significant difference from the normal group only at 72 h in both the supraspinatus and infraspinatus. In the case of the hDFs and BM-MSCs groups, macrophages were detected even after 72 h, even on day 7. Almost no macrophages were detected in the supraspinatus and infraspinatus at 24 h in all three groups, and almost no macrophage was detected even after 4 weeks. Both mast cells and macrophages were hardly detected after 4 weeks, but were found at the initial stage before day 7 B1–B4. ThThe findings based on behavioral and histological evaluation were quantified via q-PCR of nine representative genes related to inflammation, adipogenesis and muscle synthesis. The mRNA expression of inflammatory genes including TNF-a, IL-6, IL-1b, Cox2, and Ptges in the supraspinatus muscle was significantly decreased in the M-MSCs group until day 7 A1–A5. ThThere was no significant difference in the expression of the adipogenic gene, but the expression of Pparg significantly reduced in both the supraspinatus and infraspinatus muscles at 72 h. There was no difference in Myod1 in the supraspinatus, but there was a significant difference in the infraspinatus from week 4 to week 8 . M-MSCs Each cell was stained using Cell Tracker™ CM-DiI dye and then injected into the muscle during surgery. Postoperative cell duration and effects on muscle were monitored. It was confirmed that all three groups of cells remained in the muscle until 4 weeks. The number of cells remained in the order of M-MSCs, BM-MSCs, and hDFs. M-MSCs and BM-MSCs were observed intramuscularly for up to 8 weeks , and it M-MSCs are very accessible because of their unlimited source and easy production methods. Even though the cells were derived from hPSCs, M-MSCs present functional MSC features including multi-potency . In addiAdditionally, M-MSCs are relatively superior in regulating the immune response including inflammation. Previously, it was reported that the supernatants of M-MSCs contain more proteins affecting the regulation of inflammation than BM-MSCs, including (1) SMAD protein regulation and (2) negative regulation of cellular responses to oxidative stress. Secretion was confirmed.  Based onThe immunomodulatory abilities of M-MSCs were demonstrated by the transcriptomic expression levels. Compared with adult MSCs, GOs related to inflammatory inhibition and tissue recovery enhancement including GO:0050919 (negative chemotaxis), GO:0042060 , GO:0061041 , and GO:0050922 (negative regulation of chemotaxis) were upregulated in M-MSCs, while GOs related to stress reaction and inflammation including GO:0097501 , GO:0006935 (chemotaxis), and GO:1902622 (regulation of neutrophil migration) were downregulated. Based on these transcriptomic features of M-MSCs, M-MSCs are effective in regulating immune responses such as inflammation as reported previously .GO:0008330 (protein tyrosine/threonine phosphatase activity) was upregulated in M-MSCs compared with AD-MSCs, and included genes related to activities of tyrosine phosphatases and threonine phosphatases. The tyrosine phosphatases regulate the excessive activation of lymphocytes, as shown in the case of the exacerbation of acute and chronic enteritis without tyrosine phosphatases such as PTPN2 and PTPN22 in mice . In addiM-MSCs not only play an extraordinary role as cell therapeutics, but also survive for a long time based on their intensive immune evasiveness. First, M-MSCs showed an overall decrease in allograft rejection-related GOs compared with adult MSCs such as BM-MSCs and AD-MSCs at the transcriptomic level, and increased tendencies in GOs related to the negative regulation of programmed cell death. In addition, during the application of M-MSCs and adult MSCs in a mixed lymphocyte reaction (MLR) assay in which responder cells reacted with stimulator cells with mismatched histocompatibility antigens , M-MSCs The immunomodulatory ability of M-MSCs was shown to attenuate the acute inflammatory markers of damaged muscle tissue in the RCT model. When comparing the mRNA expression of inflammatory cytokines , which are mainly detected in RCT, there was a significant difference from 7 days until 4 weeks in both the upper and lower extremities. In the M-MSCs-administered group, a significantly lower or similar level of inflammation was observed compared with the golden standard BM-MSCs. In addition, it was confirmed that the group treated with M-MSCs decreased the most distressing fat infiltration in RCT compared to other groups and that myogenicity, determined by Myf5 and MyoD and acting as an indirect indicator for muscle damage caused by inflammation, decreased. It was confirmed that M-MSCs help in the recovery of the rotator cuff by reducing inflammation, preventing muscle damage, and inhibiting inflammation and fat infiltration.In the group treated with M-MSCs intramuscularly, the forelimb muscle histology and function was close to normalization. The grip strength was restored by M-MSCs to levels comparable to that of the normal group in 7 days. The BM-MSCs group showed a significant difference in the hDF group from day 7, but the normal level was attained after 8 weeks. Histological atrophy of muscle, fibrosis, and fat infiltration were confirmed via H and E staining. In the M-MSCs group, a sharp decrease was observed after 7 days, and in other cells at up to 4 weeks. The fibrosis observed via MT staining also showed a sharp decrease after day 7 in the M-MSCs group. In the case of mast cells and macrophages, a significant difference was found between the M-MSCs group and other groups. Given that these histological results and the results of forelimb grip strength are similar, M-MSCs facilitate the functional recovery of muscles from an early stage and rapid normalization. The practical clinical application of cell therapy requires the validation of safety and long-term monitoring and tracking at the cellular level. Additionally, we used rats as the RCT model, but since their recovery rate is different from humans, it is necessary to confirm whether the effect is equally potent in humans. Currently, M-MSCs are embryonic stem cell-derived cells and present an ethical problem, so their conversion to induced pluripotent stem cells is required. Therefore, we are working to construct hiPSC-derived M-MSCs. By establishing the protocol, the advantages of other adult-MSCs for culturing cells taken from patients in vitro can be obtained, and it is expected to provide a method for culturing without ethical issues.In this study, we confirmed that PSC-derived M-MSCs have superior accessibility and immunomodulatory ability and persist longer in the body compared to the conventional primary culture of adult MSCs. In addition, it was shown that the forelimb function of rats was restored in the RCT model by alleviating the acute inflammatory response by modulating the immune response. Therefore, it was confirmed that M-MSCs can be applied as a next-generation cell therapy in RCT.


Sargassum ilicifolium incorporated in an osteo-inductive scaffold comprising calcium crosslinked sodium alginate-nano hydroxyapatite-nano graphene oxide (Alg-HA-GO-F), which tends to serve as a bone graft substitute. The physiochemical characterization that includes FT-IR, XRD, and TGA confirms the structural integration between the materials. The SEM and AFM reveal highly suitable surface properties, such as porosity and nanoscale roughness. The incorporation of GO enhanced the mechanical strength of the Alg-HA-GO-F. The findings demonstrate the slower degradation and improved protein adsorption in the fucoidan-loaded scaffolds. The slow and sustained release of fucoidan in PBS for 120 h provides the developed system with an added advantage. The apatite formation ability of Alg-HA-GO-F in the SBF solution predicts the scaffold’s osteointegration and bone-bonding capability. In vitro studies using C3H10T1/2 revealed a 1.5X times greater cell proliferation in the fucoidan-loaded scaffold than in the control. Further, the results determined the augmented alkaline phosphatase and mineralization activity. The physical, structural, and enriching osteogenic potential results of Alg-HA-GO-F indicate that it can be a potential bone graft substitute for orthopedic applications.Globally, millions of bone graft procedures are being performed by clinicians annually to treat the rising prevalence of bone defects. Here, the study designed a fucoidan from  Bone defects and diseases significantly reduce the quality of human life. Bone tissue naturally possesses a good healing ability. Still, if the defect site is greater than the critical size threshold , it fails to heal on its own and needs an artificial intervention ,2. Over Alginate is a widely used natural biomaterial in tissue engineering due to its biocompatible and biodegradable properties . Its abiCarbon-based nanomaterials are gaining remarkable attention in biomedical applications due to their unique properties, including a large surface area and high mechanical potency . Carbon-Fucoidan is a sulfated polysaccharide commonly observed in the cell wall of brown algae. Fucoidan comprises fucose, glucose, galactose, mannose, xylose, rhamnose, and uronic acid ,18. SeveTo mimic the natural function of bone in terms of the porosity, osteoinduction, surface topography, and mechanical strength of the scaffold, we developed novel fucoidan-loaded Alg-HA-GO composite scaffolds for bone tissue regeneration. The scaffolds were developed by forming a calcium-crosslinked porous network of polymers via the freeze-drying method. The developed composites were examined in surface morphology, degradation, swelling behavior, and mechanical properties.Sargassum ilicifolium are shown in FT-IR analysis was performed on the developed composite scaffolds of Alg-HA, Alg-HA-GO, and Alg-HA-GO-F A. The FT−1 and 961 cm−1 assigned to the symmetric and antisymmetric stretching vibrations of P-O bonds present in the PO42− group. The bands at 599 cm−1 and 562 cm−1 correspond to the antisymmetric bending of the PO42−. The symmetric bending vibrations of the PO42− group were attributed to the bands around 467 cm−1. The characteristics peaks at 1455 cm−1 and 1416 cm−1 was for the CO32− group .The protein adsorption ability of scaffolds was measured according to the previous study . The scaThe biomineralization effect of developed scaffolds was performed using simulated body fluid (SBF). First, the SBF solution was prepared as described by Kokubo et al. . Next, t6 cells/well. The next day, the cells were treated with flakes of fabricated scaffolds. After 2 days, the media were aspirated and cells were given PBS wash, followed by the addition of chilled methanol. The plate was kept aside at room temperature for 30 min. Next, cells were stained with each dye for 15 min at 37 °C in the dark, where AO-EB stained the live and dead cells. Subsequently, fluorescence images of cells were captured using a fluorescence imager  [Fluorescent staining was performed to measure the cell viability using green dye (AO—acridine orange) and red dye (EB—ethidium bromide). The cells were seeded in the 24-well plates at a density of 0.03 × 10CA, USA) .6 cells/well were seeded in the 24-well plates and exposed to the developed scaffold. After 24 h and 48 h, media were removed, and the cells were trypsinized in order to detach. Then, the collected cells were suspended with an equal volume of media. Further, the cells were counted using a haemo-cytometer by mixing the cell suspension with 0.4% trypan blue dye in a 1:1 ratio.To study the cell proliferative effect, cells at a density of 0.03 × 10Scanning electron microscopy assessed the cell adhesion and morphology studies of the cell scaffold. Initially, composites were immersed in the media overnight, followed by cell seeding. After two days of culture, the cell-distributed scaffolds were given PBS wash and fixed using formaldehyde. After, dehydration with ethanol at increasing concentrations  for 3 min was repeated three times in each solution. Finally, the cell–scaffold constructs were air-dried overnight before the assessment under SEM  .6 cells/well was cultured in 24-well plates. The confluent cells were treated with composite scaffold Alg-HA, Alg-HA-GO, and Alg-HA-GO-F for 7 and 14 days in an osteogenic differentiation medium. The media were changed every two days. After incubation, the cells were washed with 1X PBS twice, followed by homogenization of the cells using 100 µL 25 mM carbonate buffer containing 0.2% Triton X-100 (pH-10.3). Further, 50 µL 250 mM carbonate buffer containing 2.5 mM MgCl2 and 15 mM p-nitrophenyl phosphate was added. Soon after, the plate was incubated at 37 °C for 30 min, and 1 M NaOH was used to stop the reaction. The absorbance of the reaction was measured at 405 nm. The cells cultured in osteogenic differentiation media were taken as control.The cell density of 0.03 × 106 cells/well were seeded in a 24-well plate and cultured with scaffolds for 14 and 21 days. After 14 and 21 days, cells were fixed with 4% paraformaldehyde for 30 min and stained with 40 mM alizarin red s solution. After 30 min of incubation, the calcium-stained cells were imaged using a microscope . Further, 10% cetylpyridium chloride in 10 mM sodium phosphate solubilized the cells for quantification. After incubation at 37 °C for one hour, the absorbance was measured at 570 nm.Alizarin red-s and Von Kossa staining scrutinized the calcium deposition. The murine MSCs at a density of 0.03 × 10In addition, Von Kossa staining measured the mineralization. After 14 days of culture, cells were fixed using 4% paraformaldehyde, and 1% silver nitrate solution was added. Subsequently, the plate was exposed to light for 30 min, and the cells were rinsed with distilled water after adding 5% sodium thiosulfate for two minutes. After alcohol wash, the formation of black deposits was observed .All of the data are expressed as means ± the standard deviation of a minimum of three replicates for each composite in every experiment, using Origin Lab  and GraphPad Prism 8.0. We developed fucoidan-loaded Alg-HA-GO osteoinductive scaffolds for bone tissue repair and regeneration. The FT-IR confirms the ionic interaction between the materials, the XRD shows the sharp crystalline peaks of HA, and the other materials used are amorphous. The surface properties analyzed by FESEM and AFM reveal that the pore size between 96–460 μm and has 426–260 nm nanoscale roughness from all of the composites. The slower rate of biodegradation and increased protein adsorption of Alg-HA-GO-F compared to Alg-HA and Alg-HA-GO are suitable for bone regeneration. The slow and sustained release of fucoidan positively influences osteogenic differentiation. The composites can form biomineralization using SBF. Porous surfaces with nanoscale topography stimulate the cell attachment, proliferation, and differentiation of stem cells. The in vitro studies of scaffolds showed an enhanced murine MSCs proliferation, alkaline phosphatase activity, and calcium deposition. The fucoidan containing alginate, hydroxyapatite, and graphene oxide will be excellent biomaterial for orthopedic applications.


It is specifically designed to assist researchers pursuing studies in metabolomics, exposomics and analytical chemistry. More specifically, CFM-ID 4.0 supports the: 1) prediction of electrospray ionization quadrupole time-of-flight tandem mass spectra (ESI-QTOF-MS/MS) for small molecules over multiple collision energies ; 2) annotation of ESI-QTOF-MS/MS spectra given the structure of the compound; and 3) identification of a small molecule that generated a given ESI-QTOF-MS/MS spectrum at one or more collision energies. The CFM-ID 4.0 web server makes use of a substantially improved MS fragmentation algorithm, a much larger database of experimental and in silico predicted MS/MS spectra and improved scoring methods to offer more accurate MS/MS spectral prediction and MS/MS-based compound identification. Compared to earlier versions of CFM-ID, this new version has an MS/MS spectral prediction performance that is ∼22% better and a compound identification accuracy that is ∼35% better on a standard (CASMI 2016) testing dataset. CFM-ID 4.0 also features a neutral loss function that allows users to identify similar or substituent compounds where no match can be found using CFM-ID’s regular MS/MS-to-compound identification utility. Finally, the CFM-ID 4.0 web server now offers a much more refined user interface that is easier to use, supports molecular formula identification (from MS/MS data), provides more interactively viewable data (including proposed fragment ion structures) and displays MS mirror plots for comparing predicted with observed MS/MS spectra. These improvements should make CFM-ID 4.0 much more useful to the community and should make small molecule identification much easier, faster, and more accurate.The CFM-ID 4.0 web server (  IncreEU/Japan ,8 and GNEU/Japan , these dEU/Japan  or METLIEU/Japan ,14, but in silico methods. In-silico MS-based compound identification methods were developed to help researchers identify compounds from an experimentally collected MS/MS spectrum without directly needing or querying an experimentally collected reference MS/MS spectral database. State-of-the-art methods for in silico MS-based compound identification use a wide array of different techniques, ranging from MS/MS spectral prediction to MS/MS spectral fingerprint analysis. Nearly all of these methods employ combinations of rule-based expert systems and the latest deep learning methods  is an example of an  in 2014 ,33. Unli in 2014  and CSI: in 2014 , CFM-ID f CFM-ID , this mef CFM-ID  and thenf CFM-ID . Since iin silico and experimental MS/MS databases, enhancements to the MS/MS spectral displays and support for neutral loss spectral searching were all deemed to be essential to maintain CFM-ID’s relevance to the user community. This paper describes these upgrades and updates, by formally introducing the CFM-ID 4.0 web server. This paper also demonstrates how these enhancements have improved the overall accuracy and performance of CFM-ID relative to earlier versions and relative to competing software tools.While CFM-ID remains very popular, a number of its algorithms, its performance and its visual displays have become somewhat dated. This motivated us to start upgrading both the back-end and the front-end of the CFM-ID server. For instance, recent developments in machine learning along with the availability of an expanded MS/MS training set allowed us to substantially improve the performance of the fragmentation modeling in the CFM-ID algorithm . Such a Spectra Prediction, Peak Assignment, and Compound Identification.The CFM-ID 4.0 web server offers three general functions: 1) predicting electrospray ionization quadrupole time-of-flight tandem mass spectra (ESI-QTOF-MS/MS) for chemical compounds over multiple CID energies (C2MS); 2) annotating ESI-QTOF-MS/MS spectra given the structure of the parent compound; and 3) identifying the chemical compound that produced the given centroided ESI-QTOF-MS/MS spectra (MS2C). These three functions are listed on the CFM-ID home page as: Spectra Prediction utility performs the C2MS operation. To use this function, a user must enter the structure of a neutral compound using either SMILES . The output of the Spectra Prediction function is presented in two different formats: (1) a human-readable, downloadable text file containing the predicted MS/MS spectrum, and (2) an interactive image of the predicted MS/MS spectrum. Each displayed peak in the interactive spectral display includes a high precision m/z value, a relative intensity value, and an image of the most likely associated fragment ion structure. This information can be viewed by mousing-over each spectral peak. In addition to generating a predicted in-silico MS/MS spectrum, CFM-ID 4.0 will also retrieve the experimentally measured MS/MS spectrum, if such a spectrum exists in the CFM-ID experimental spectral library. This experimental MS/MS spectral library contains both internally collected and externally collected MS/MS spectra provided by MoNA  structure. This utility is intended to help improve the explainability of product ion MS/MS spectra and has been widely used as a teaching tool. For a given MS/MS spectrum and the corresponding (known) chemical structure, the Peak Assignment tool attempts to assign a possible fragment ion to each peak in the MS/MS spectrum. To use the Peak Assignment utility, a user must submit the known compound structure and a corresponding list of m/z peaks from an ESI-MS/MS experiment. Users must also select the corresponding charge type, adduct type, and mass tolerance value. The mass tolerance value (default to 10 ppm) is the tolerance used to match the observed MS/MS peaks to the predicted fragment ions calculated by CFM-ID 4.0. As with the Spectra Prediction utility, the output for Peak Assignment is displayed in a color-coded mass spectrogram, where annotated peaks are marked in red and unannotated peaks (if any) in blue. In addition to generating an interactively viewable MS/MS spectrum, where fragment ion structures can be viewed by mousing-over a given peak, the user can also download an additional text file. This file contains the annotated peak list and the corresponding SMILES strings for the identified ion fragments.CFM-ID 4.0’s Compound Identification utility supports its MS2C operations. In particular, it allows users to identify metabolites from one or more user-supplied experimental MS/MS spectra. CFM-ID 4.0 offers users two options: 1) Regular Compound Identification via product ion MS/MS spectra and 2) compound identification via Neutral Loss Search  pure compound collected at one or more specified collision energies: low (10 eV), medium (20 eV), and/or high (40 eV) – either entered directly in text boxes or uploaded as files. Users must also supply the desired spectral type (only ESI is offered in version 4.0), the ion mode (positive or negative), the adduct type (the parent ion adduct), the parent ion mass (measured from the ion selection filter to collect the MS/MS spectrum), the candidate mass tolerance, the scoring function , the number of results to be viewed and the mass tolerance for peak matching in the spectral display (default to 10 ppm). Users must also select from a set of 18 databases carefully curated databases containing experimental and/or in silico-predicted MS/MS spectra along with their corresponding compound structures. Once these data are submitted, by pressing the Submit button, it typically takes a less than a minute for the CFM-ID server to return a sorted list of possible compound structures. The amount of time taken depends on the number and size of the spectral databases being searched. While compound structures can be identified with only a single MS/MS spectrum collected at a single collision energy level, providing spectra at two or more energy levels will certainly improve the identification accuracy. Users can specify which of three methods for scoring the similarity between observed versus predicted MS/MS spectra to use: 1) the Dice score, 2) the dot product score, and 3) the dot product + Metadata score. Details of the Dice score and dot product ranking methods can be found in reference  of a smaller ‘substructure’ such as a water molecule, an ammonia molecule, a phosphate group, a sugar group or some other minor chemical modification. Normally small m/z shifts arising from minor chemical modifications make it impossible for conventional MS/MS spectral searching operations to identify chemically modified or chemically similar molecules. On the other hand, neutral loss searching allows these minor modifications to be ignored during the spectral searching process. In this way CFM-ID’s Neutral Loss Search allows users to identify chemical compounds that are chemically similar to chemical compounds in the CFM-ID 4.0 spectral databases but which have no actual structure or actual product ion MS/MS spectra in the CFM-ID 4.0 databases. An illustrative example of a Neutral Loss Search using CFM-ID 4.0 is shown in Figure Compound Identification search with an experimentally measured MS/MS spectrum of warfarin (where the MS/MS spectrum of warfarin has been deliberately removed from the selected CFM-ID database) yields no significant, or chemically similar matches. On the other hand, a Neutral Loss Search of the same compound that uses the automatically calculated neutral loss spectrum of warfarin against CFM-ID’s neutral loss MS/MS database identifies one molecule that is almost chemically identical to warfarin as its top hit. Similar to other interactive spectral viewers in CFM-ID, mousing over the neutral loss ions allows users to identify the structures of many of the substituent ions. These high scoring hits and the identification of key neutral loss ions off the opportunity for users to determine the approximate structure of hitherto unknown compounds or compounds not in the CFM-ID spectral databases.In contrast to the regular Neutral Loss Search  for the server to complete the calculation and to sort the hits. The Neutral Loss Search provides two example spectra (Example #1 and #2) for users to try.To identity or partially identify a structure via the s Search , users mNeutral Loss Search and regular Compound Identification function is presented in two parts: an MS/MS mirror plot for comparing spectra and a tabular list of ranked compounds. An example of such an output can be found in Figure Compound Identification results.The output of both the Help section, and most importantly, offering a better interactive display of the MS/MS spectra. More specifically, we redesigned CFM-ID 4.0’s Home Page, so that it now shares the same layout and styling with our other popular databases and web servers such as HMDB 5.0 . While the MSRB algorithm is faster than the machine learned algorithm, it can only compute MS/MS spectra for compounds from a relatively small set of chemical classes, including lipids, polyphenols, acylcarnitines and acylglycines  to assess its prediction performance. Compared to the CFM-ID 2.0 and the CFM-ID 3.0 web servers, CFM-ID 4.0 was able to predict MS/MS spectral significantly more accurately. As also discussed in  dot product score of 0.38 and 0.35 for [M + H] + and [M-H]- spectra, respectively. This corresponds to a ∼26% and a ∼21% performance gain compared to CFM-ID 3.0. Figure The changes introduced to CFM-ID 4.0 necessitated a careful evaluation of its prediction performance to ensure that these improvements were robust and significant. As described in , we perfussed in , the preCompound Identification). As described in  value, then comparing this Orbitrap HCD spectrum with the CFM-ID-predicted spectrum with a CID energy that is closest to the NCE . As noted previously in  performance to SIRIUS 4 (in silico HMDB 5.0 spectra) outperformed SIRIUS 4 in terms of chemical compound identification accuracy for the positive ion mode, while SIRIUS 4 showed a slight advantage in the negative ion mode (for the top ranked hits). Interestingly, SIRIUS 4 could only identify chemical compounds from a given MS/MS spectrum in 93 out of 108 cases. There were 15 cases where no chemical structure was produced, and 4 out of these 15 cases proposed no chemical formula.Although CFM-ID 4.0 is by no means perfect in identifying compounds purely from their MS/MS spectra, it is almost perfect at determining the correct chemical formula. As shown in Table SIRIUS 4  using HMhttps://hub.docker.com/r/wishartlab/cfmid). This docker image only contains the core functionality of CFM-ID and does not include any pre-computed MS/MS spectral data. More information is available in CFM-ID’s Help section about this core image. However, the downloadable version of CFM-ID 4.0 enables large-scale computation or the processing of sensitive data, which cannot easily be supported on a publicly accessible server. The CFM-ID server is hosted on a quad-core (Intel Xeon 8175M) virtual server with 8G of RAM. The current configuration has a three minute time-out on MS/MS spectral prediction tasks. The amount of time required to perform a spectral prediction is largely dependent on the molecular weight (MW) and the number of bonds in the molecule. Typically, a molecule with a MW < 1000 Da will take less than one minute to predict.The CFM-ID 4.0 web server is organized into two components: 1) the web layer that serves all the web pages and handles data storage and 2) the computational core, which handles all of the spectral predictions and calculations. The web layer was developed using the Ruby on Rails framework (version 5). Ruby on Rails is a development system that employs the Model-View-Controller (MVC) concept, where models respond and interact with the data, views create the interface to show and interact with the data, and controllers connect the user to the views. This framework allowed the CFM-ID 4.0 programming team to rapidly develop, prototype and test all CFM-ID’s web modules and page views. MySQL and Redis were used for the back-end and HTML, CSS, JavaScript, and the D3.js library were used for the front-end. The computational core consists of several specialized CFM-ID 4.0 algorithms developed in C++ and Java, including the machine-learned models and the rule-based extension implementations (CFM-ID MSML 4.4.5 and CFM-ID MSRB 1.1.13). The machine-learned models deployed on the CFM-ID 4.0 web server were all derived from the recently published updates to the CFM-ID algorithm . Each coin-silico MS/MS spectra, a greatly expanded CFM-ID spectra library, and the introduction of a Neutral Loss Search feature, we believe the CFM-ID 4.0 web server will be much more useful to users wishing to perform MS2C tasks for identifying known structures or those wishing to identify completely novel structures. While the improvements to both front-end and back-end are significant, we are still planning to implement several extensions to the CFM-ID 4.0 web server over the coming year. In particular, we expect to re-introduce a much-improved EI-MS spectra prediction module and a significantly improved EI-MS compound identification module (for GC-MS-based metabolomics) that will support user input of experimentally measured retention indices and EI-MS spectra. We also plan to extend the next version of CFM-ID to support Orbitrap MS/MS spectral predictions. These additions will likely be introduced in later 2022 or early 2023. Overall, we believe the improvements already described here along with the planned improvements to the server's functionality will make CFM-ID 4.0 much more useful to the analytical chemistry community and should make small molecule identification easier, faster and more precise.The CFM-ID 4.0 web server offers a suite of utilities to facilitate automated MS/MS spectral prediction (C2MS), spectral annotation and chemical compound identification (MS2C). Compared to previous versions, the CFM-ID 4.0 web server is more user-friendly, more accurate and more informative. In particular, CFM-ID 4.0’s functional improvements include an improved user interface, a new suite of neutral loss searches and neutral loss annotations, improved spectral displays (mirror plots), more informative data tables, improved prediction capabilities, enhanced documentation and greater user-friendliness. CFM-ID 4.0’s C2MS performance has been thoroughly benchmarked and proven to be significantly better than previous versions . Its MS2


This study aimed to develop a miltefosine-eluting contact lens (MLF-CL) device that would allow sustained and localized miltefosine release for the treatment of Acanthamoeba keratitis. MLF-CLs were produced in three different miltefosine doses by solvent-casting a thin miltefosine-polymer film around the periphery of a methafilcon hydrogel, which was then lathed into a contact lens. During seven days of in vitro testing, all three formulations demonstrated sustained release from the lens at theoretically therapeutic levels. Based on the physicochemical characterization of MLF-CLs, MLF-CL’s physical properties are not significantly different from commercial contact lenses in terms of light transmittance, water content and wettability. MLF-CLs possessed a slight reduction in compression modulus that was attributed to the inclusion of polymer-drug films but still remain within the optimal range of soft contact lenses. In cytotoxicity studies, MLF-CL indicated up to 91% viability, which decreased proportionally as miltefosine loading increased. A three-day biocompatibility test on New Zealand White rabbits revealed no impact of MLF-CLs on the corneal tissue. The MLF-CLs provided sustained in vitro release of miltefosine for a week while maintaining comparable physical features to a commercial contact lens. MLF-CL has a promising potential to be used as a successful treatment method for Acanthamoeba keratitis. Acanthamoeba keratitis (AK) is a rare but potentially blinding corneal infection caused by free-living Acanthamoeba amoebae ,2. InadeMiltefosine is an alkylphosphocholine agent with a broad spectrum of antiparasitic properties, which has recently been used in AK. This drug is commonly used as an oral medication to treat leishmaniasis . After oPreviously, we have described a contact lens drug delivery system involving the incorporation of a thin drug-poly[lactic-co-glycolic acid] (PLGA) film into a poly[hydroxyethyl methacrylate] (pHEMA) hydrogel. This system has previously been used to achieve zero-order release kinetics at therapeutically relevant concentrations with fluorescein and ciprofloxacin for up to one month .In this study, we incorporated miltefosine into this contact lens system to create a therapeutic contact lens (MLF-CL) for the treatment of AK. Miltefosine was loaded at different concentrations and the physicomechanical properties  of the MLF-CLs were investigated and compared with commercial contact lenses. Drug release kinetics were measured for a period of one week. In addition, the cytocompatibility and in vivo biocompatibility of the MLF-CL was evaluated.Miltefosine was purchased from Cayman Chemical . Liquid methafilcon and methafilcon contact lens blanks were purchased from Kontur Kontact Lens . Poly(lactide-co-glycolide), 85% lactide, 15% glycolide (MW 190 KDa) was purchased from Merck and Cie . Hexafluoroisopropanol (HFIP) was purchased from Oakwood Chemical . Gibco phosphate-buffered saline (PBS), Dulbecco’s Modification of Eagle’s Medium, penicillin-streptomycin, and L-glutamine were purchased from Thermo Fisher Scientific . Biopsy punches were purchased from Sklar Instruments . All other reagents were purchased from Millipore-Sigma , except where otherwise noted.® 2959  and 3.5 min of UV light exposure at 120 mW/cm2 applied within a Zeta 7401 UV chamber  for 3.5 min at 120 mW/cm2. The methafilcon cylinder was then lathed into a contact lens that consisted of the drug-PLGA film fully encapsulated within methafilcon, with the initial cylinder on the front curve and polymerized methafilcon on the back curve  to assess the morphology. Non-hydrated MLF-CLs were positioned with the convex side of the lens facing the OCT camera. Raster scanning imaging was used in four segments for each contact lens to obtain cross sectional images of the contact lens and drug-polymer film.In vitro release of the MLF-CLs was conducted by hydrating MLF-CLs in 5 mL phosphate buffered saline (PBS) and placing in an incubator shaker at 37 °C at 64 rpm. At predetermined time points, the MLF-CLs were removed from PBS and immersed in fresh PBS. Release buffer aliquots were stored at 4 °C until drug concentration was quantified. Four lenses were used per formulation. Release quantification was performed by a colorimetric method.sW = weight at equilibrium swelling and dW = dry weight.Water content of the MLF-CLs was performed by gravimetric method, according to ISO-18369-1 guidelines . MLF-CLsLight transmission was measured through commercial contact lenses and the central aperture of the hydrated MLF-CL using SpectraMax M3 . The aven = 4 and 5 measurements per specimen) was determined using ImageJ software  as a function of time.MLF-CLs were hydrated in PBS until they reached equilibrium swelling. A drop of distilled water (about 5 µL) onto the surface via a syringe. Then, a high-resolution image was captured from the side using the Dino-Lite Edge camera. The contact angle for each formulation . Before analysis, samples were hydrated by immersing them in PBS for 2 days. The compression test was conducted on rings with 7 mm and 6 mm external and internal diameter, respectively and with a crosshead speed of 1 mm/min. The Compression Modulus was obtained from the linear derivative of the stress–strain curve in the low stiffness range (less than 10% strain). Three independent measurements were conducted for each group.5 cells/mL the day prior. The cells with extract were incubated for 24 h. Cell viability was measured by CellTiter 96® Non-Radioactive Cell Proliferation Assay (MTT) . Results were expressed as percentage viability of L929 controls that were not exposed to extract. Four lenses were used for each formulation, with six replicates per lens.L929 murine fibroblasts  were used to assess the cytotoxicity of MLF-CLs. L929 cells were grown in Dulbecco’s Modification of Eagle’s Medium (DMEM), supplemented with 10% fetal bovine serum, 1% penicillin-streptomycin, and 4 mM L-glutamine. Cytotoxicity was measured by the minimum elution media method, described by ISO 10993-5 guidelines . MLF-CLsThe study protocol #2021N000159 was approved on 7 July 2021 by the Institutional Animal Care and Utilization Committee at Schepens Eye Research Institute of Massachusetts Eye and Ear Infirmary . All animals were treated according to the Association for Research in Vision and Ophthalmology (ARVO) Statement for the Use of Animals in Ophthalmic and Vision Research . New Zealand white rabbits (NZW)  age four months, each weighing 3–5 kg, were used for the biocompatibility study. Only one eye in each NZW was used to prevent bilateral vision restriction. The contralateral eye was untreated and served as a control. NZWs were singly housed in a climate-controlled environment with free access to food and water during acclimation and throughout the experiment. NZWs were acclimated for seven days prior to experiments. In each of the studies, intramuscular injection of 30 mg/kg ketamine, 5 mg/kg xylazine, and 1 mg/kg acepromazine were used for anesthesia. 0.05 mg/kg Buprenorphine HCl was used for pain control prior to tarsorrhaphy.4 was used for the biocompatibility study. Under anesthesia, NZWs had baseline photos taken by a Topcon DC-3 digital camera attachment for slit lamp. The MLF-CL was placed on the cornea, with care to place it under the nictating membrane. The MLF-CL was evaluated to ensure free movement on the cornea without fluting or bubbling. To improve lens retention, eyes with contact lenses received a temporary tarsorrhaphy in which the eyelids were closed with one to two 5-0 Nylon sutures. NZWs were evaluated daily for signs of discomfort. After three days, the tarsorrhaphy and MLF-CL and re-imaged. NZW eyes were evaluated for redness, edema, discharge, and fur matting. NZWs were euthanized with 120 mg/kg pentobarbital, and the corneas were removed from both eyes. The eyes were fixed in 10% formalin, embedded in paraffin, and cut into 10-micron sections in an anterior to posterior fashion so that the pupil and the optic nerve were in one section (PO section). Slides were stained with hematoxylin and eosin (H&E) to visualize ocular structures.Only MLF-CLp-Value of less than 0.05 was considered significant.For all continuous data, nonparametric descriptive statistics were used to determine statistical significance. Kruskal–Wallis Test was calculated, and if significant, followed by pairwise comparisons to determine significant differences between groups. A 2: 83.30 ± 6.08 μm, MLF-CL4: 81.11 ± 1.15 μm, MLF-CL8: 85.03 ± 5.62 μm) . When imaged by OCT, no delamination of the film from the hydrogel was observed before hydration or after the lens was hydrated  to 470.3 ± 39.8 µg (MLF-CL8)  values and the largest Model Selection Criterion (MSC) [Drug release from MLF-CLs was studied in PBS at 37 °C. Because miltefosine aqueous solubility is relatively high (reported as 440 mg/mL ), frequeMLF-CL8) . We analMLF-CL8) . Consideon (MSC) , the relon (MSC) ,35.n = 4/group). Commercial contact lens had a water content (53 ± 1.6%) comparable to the reported value (55%) [p = 0.49, Kruskal–Wallis Test) difference in the water content between MLF-CLs and commercial methafilcon lenses  ,40. Thern lenses .p = 0.15, Kruskal–Wallis Test, n = 4 per group, The light transmittance properties of contact lenses are clearly important for visual performance. MLF-CL maintained light transmittance >95% through the central aperture between 390 nm to 700 nm A. The din = 3/group, 2 (220 ± 50 kPa), MLF-CL4 (296 ± 92 kPa) and MLF-CL8 (255 ± 30 kPa) with no significant differences among them .Excessive stiffness in a contact lens can result in discomfort. Compression tests  were perp = 0.06, Kruskal–Wallis Test) among the MLF-CL groups (n = 4/group)  .2, MLF-CL4, and MLF-CL8, respectively  wore the MLF-CL4s continuously for three days. One eye showed erythema throughout the conjunctiva, on day 3. In the other two rabbits, no abnormalities were seen in daily observations during the lens wear period. No erythema, discharge, lid edema, or epithelial defects were noted on slit lamp exam after three days of continuous wear.Potential risks with contact lens wear include corneal edema, cornea abrasions, and microbial keratitis and there is also the risk of local or systemic toxicity from topically delivered miltefosine. Normal New Zealand White (NZW) rabbits were used to assess biocompatibility as rabbits are the lowest order mammal that can wear a contact lens. In addition, NZW eye dimensions are comparable to those of a human . MLF-CL4Pathology revealed normal corneas and irises in all NZWs. No changes in the corneal epithelium, stromal lamellae, Descemet’s membrane, and endothelium were observed . ResultsIn this work, we have encapsulated miltefosine in a contact lens hydrogel. This device provides sustained release of miltefosine for one week and may be of use in the treatment of AK. To our knowledge, this is the first report of a miltefosine sustained release system for the treatment of ocular disease.PLGA was chosen due to its safety and excellent sustained drug release profiles . PLGA isIn a miltefosine-loaded nanostructured lipid carriers release system , most dr2, MLF-CL4 and MLF-CL8 released 24, 85 and 151 µg of miltefosine on the first day, respectively. The daily release rate thereafter ranged from 17-26 μg/day for MLF-CL2, 24–42 μg/day for MLF-CL4, and ranged from 41-89 μg/day for MLF-CL8  of 16 µg/mL for clinical and environmental isolates ,60,61. T MLF-CL8 B after oThe light transmittance of central aperture was above 95% for all three formulation and was comparable to commercial lens, though the film-embedded part of the MLF-CL showed semitransparency . We alsoWettability and mechanical properties of the lenses are critical for the physiological compatibility, handling, durability, and comfort of wearing the lenses. In fact, oxygen diffusivity and water content are key parameters influencing the comfort and safety of a lens . MLF-CLsThe modulus range for the MLF-CLs we measured was between 210 kPa and 300 kPa. We found that MLF-CLs are softer than commercial contact lenses (490 kPa) as they showed a significantly lower modulus. This decrease in modulus is most likely the result of increased chain mobility in the contact lens hydrogel. Nonetheless, the modulus of MLF-CL is still within the acceptable range of values reported in the literature for the currently available contact lens materials (300 to 600 kPa) .2 had the highest cell viability (91%) and MLF-CL8 had the lowest cell viability (36%). Given that the vehicle lens had cell viability of close to 100%, the decreased cell viability is solely associated with increased miltefosine dose. Nonetheless, MLF-CL4 had an acceptable level (73%) of cell survival by ISO standards [4, combined with minor in vivo toxicity, additional formulations can be investigated with changing miltefosine concentrations to reduce cytotoxicity. We also have the flexibility to adjust the MLF-CL central aperture size to achieve this purpose.MLF-CL showed cell viability inversely proportional to dose: MLF-CLtandards . In additandards . In our In the biocompatibility experiment for this study, one of three rabbits exhibited adverse effects after three days of wear. Miltefosine acts as an immunomodulator, activating Type 1 T-helper cells (Th1) which in turn increase the recipient’s inflammatory response. Cytokines, particularly interferon gamma and interleukin 12 have been observed to be elevated with oral miltefosine use . However4 lens is very suitable for further in vivo and clinical studies, as it shows comparable physicomechanical properties to the commercial contact lens, optimal biocompatibility, and an enhanced and sustained drug release over a period on one week.We incorporated the miltefosine-PLGA film into a commercially available hydrogel material to form a delivery system that can slowly release miltefosine. MLF-CL’s physical properties were not significantly different from commercial contact lenses in terms of light transmittance, water content, and wettability. This ocular drug delivery system provides a slow and steady release of miltefosine over the course of a week and has the safety profile to serve as a promising drug treatment platform for AK. More critical efforts are needed to develop an efficient and safe MLF-CL device that can be validated in animals and clinics. In accordance with the outcomes of this investigation, we envision that the MLF-CL


The as-obtained C460@Tb-UiO-66-(COOH)2 samples showed highly selective recognition, excellent anti-interference and rapid response characteristics for the recognition of TDGA. The detection limit is 0.518 mg·mL−1, which is much lower than the threshold of 20 mg·mL−1 for a healthy person. In addition, the mechanism of TDGA-induced fluorescence quenching is discussed in detail. This sensor is expected to detect TDGA content in human urine.Thiodiglycolic acid (TDGA) is a biomarker for monitoring vinyl chloride exposure. Exploring a facile, rapid and precise analysis technology to quantify TDGA is of great significance. In this research, we demonstrate a fluorescent sensor based on dual-emissive UiO-66 for TDGA detection. This ratiometric fluorescent material named C460@Tb-UiO-66-(COOH) Vinyl chloride (VCM) is an important industrial chemical used to produce cables, pipes and household equipment ,2. HowevIn recent years, fluorescence detection has received great attention owing to its facile operation, low instrument cost, fast response and high sensitivity ,14. Lumi2 with isomeric ligand extension was synthesized firstly. Then, a new ratiometric fluorescent material C460@Tb-UiO-66-(COOH)2 was designed and synthesized by simultaneous introducing the organic dye  and Tb3+ into the pores of UiO-66-(COOH)2. It was found that the C460@Tb-UiO-66-(COOH)2 samples with double blue-green emission have the advantages of excellent selectivity, high sensitivity, and rapid response for detection Thiodiglycolic acid (TDGA) in urine. In addition, fluorescent test paper was successfully prepared for the rapid detection of TDGA. The color changes visible to the naked eye were presented. This functionalized UiO-66 material is expected to be an ideal sensor for TDGA detection.In this work, UiO-66-(COOH)All chemicals used in this experiment were analytically pure and used without purification. All measurements were performed at room temperature.2 was synthesized using a hydrothermal method with some modifications  + 1.76624 and the correlation coefficient (R2) is 0.9738, where [C] presents the concentration of TDGA. According to three IUPAC criteria, the limit of detection (LOD) is calculated by the following two equations:Sb is the standard deviation; N is the number of blank (n = 21); F0 is the I544 nm/I420 nm of C460@Tb-UiO-66-(COOH)2 in water; F1 is the mean of F0; S is the slope of the linear relationship in −1, which is far below the threshold value of 20 mg·mL−1 in healthy individuals [Sensitivity is a major factor in determining the application of a sensor in real-life applications. Concentration-dependent experiments were further investigated to evaluate the sensitivity. The sensitivity experiment was performed by measuring the corresponding fluorescence intensity with different concentrations of the TDGA in the aqueous solutions. There is a linear relationship between them in the concentration range from 0 to 0.01 M . The linividuals .2 has been further investigated in detail. Generally, the mechanism for the detection can be attributed to three reasons [2; (2) the interaction between the lanthanide ions and TDGA; (3) the interaction between ligands and TDGA. To reveal the possible sensing mechanisms, XRD analysis was first carried out  reasons : (1) theried out . After a2, revealing that the energy transfer between the ligand and TDGA is inefficient and it can-not actually affect the luminescence efficiency of the Tb3+ [In the abstract, the luminescence efficiency of LMOFs depends on the energy transfer between the metal and organic ligand . In genethe Tb3+ .2 is based on a static mechanism or dynamic mechanism. Currently, the static mechanism is due to the formation of non-emitting intermediates between the fluorophore and the quenching agent, while the dynamic quenching is attributed to the collision between the excited fluorophore and the quenching agent [2 monitored at 420 nm is almost unchanged (from 0.0051 μs to 0.0050 μs). However, the fluorescence lifetime of C460@Tb-UiO-66-(COOH)2 monitored at 544 nm is significantly reduced from 802.2 μs to 21.992 μs. It indicates that the fluorescence quenching in this study is mainly caused by the interaction between terbium ion and TDGA, which belongs to dynamic quenching.Fluorescence lifetime is a very important parameter to judge the fluorescence quenching mechanism, which could usually reflect whether the quenching effect of TDGA to C460@Tb-UiO-66-(COOH)ng agent . Therefo2 with blue-green dual emission has been fabricated and developed as an excellent fluorescence analysis probe for the sensing of TDGA. Aqueous suspensions of C460 and Tb-UiO-66-(COOH)2 simultaneously generate the characteristic emission of lanthanide ions and dye with a single excitation mode. It was found that C460@Tb-UiO-66-(COOH)2 can be used as a fluorescent probe for TDGA recognition. This probe has the advantages of excellent selectivity, good sensitivity and fast response. The calculated detection limit is much lower than the threshold value in healthy subjects. In addition, a portable C460@Tb-UiO-66-(COOH)2 test paper was prepared. Based on this, C460@Tb-UiO-66-(COOH)2 is expected to be a fluorescent sensor to detect TDGA in urine.In conclusion, a ratiometric fluorescent probe C460@Tb-UiO-66-(COOH)


Although the root-mean-square roughness was reduced from 2.00 to 0.98 nm, the surface was still extremely rough for direct bonding. In the second approach, an Au/Ti/Au multilayer on a smooth SiO2 template is bonded with a packaging substrate, and the Au/SiO2 interface is exfoliated. The transferred Au/Ti/Au getter layer has a smooth surface with the root-mean-square roughness of 0.54 nm and could form wafer-scale direct bonding at room temperature. We believe that the second approach would allow a simple packaging process using direct bonding of the getter layer.This study demonstrates room-temperature bonding using a getter layer for the vacuum packaging of microsystems. A thick Ti layer covered with an Au layer is utilized as a getter layer because it can absorb gas molecules in the package. Additionally, smooth Au surfaces can form direct bonds for hermetic sealing at room temperature. Direct bonding using a getter layer can simplify the vacuum packaging process; however, typical getter layers are rough in bonding formation. This study demonstrates two fabrication techniques for smooth getter layers. In the first approach, the Au/Ti layer is bonded to an Au layer on a smooth SiO Several types of micro-electro-mechanical systems (MEMS) are degraded by gas molecules in packages. Thus, the vacuum packaging process is an important step in the performance and lifetime of MEMS devices. Low-temperature bonding techniques have been extensively studied for the packaging of MEMS assemblies to suppress thermal damage ,4,5,6,7.Additionally, it is known that the Au/Ti layer can function as a non-evaporative getter (NEG), which absorbs residual gas molecules in the package . When thStudies on direct bonding typically utilize thin Au/Ti layers, such as sputter-deposited Au and Ti layers with thicknesses of 15 and 3 nm, respectively . The preOne possible solution is the use of chemical mechanical polishing (CMP) on a thick Au/Ti film; however, a chemically stable Au surface has difficulty in CMP planarization . Another2 layer was fabricated on the Si substrates in a thermal oxidation furnace. This is because the oxidized Si substrate has an atomically smooth surface and the Au film badly adheres to the SiO2 surface. Thus, a smooth Au surface can be fabricated via mechanical exfoliation at the Au/SiO2 interface. The fabricated smooth getter film was directly bonded to an Au/Ti-metalized Si substrate to demonstrate the packaging process. Atomic bonds between the Au surfaces were generated using a surface-activated bonding method; the Au surfaces were cleaned with Ar plasma and thereafter contacted with each other at room temperature. The plasma was activated at 200 W for 30 s in pure Ar gas at a pressure of 60 Pa. After the bonding step, a 100-µm-thick razor blade was inserted between the bonded wafers. This causes the exfoliation of wafers at the Au/SiO2 interface.In the present study, smooth getter films were fabricated by designing a TS process, and the smooth film was directly bonded to another substrate at room temperature. The Au and Ti layers were deposited using a sputter deposition machine . The metal films were formed on oxidized 4-inch-diameter Si substrates; a 300-nm-thick SiOThe surface morphology of the fabricated getter film was evaluated using atomic force microscopy (AFM). The quality of the direct bonding of the smooth surface to another substrate was evaluated by scanning acoustic microscopy (SAM) and a tensile tester. Additionally, the nanostructure of the bonding interface was observed using transmission electron microscopy (TEM).In this study, two TS-based processes for the fabrication of smooth getter films were demonstrated for wafer-level packaging via room-temperature direct bonding.2 template; subsequently, a 6-nm-thick Au layer covered the Ti layer. The Au/Ti getter layer had a relatively rough surface for the formation of direct bonding. The Au/Ti layer was bonded with a 20-nm-thick Au thin film deposited on a SiO2 template, which has an atomically smooth surface with an RMS roughness of ~0.2 nm, at 200 °C under a bonding load of 1.27 MPa (10 kN for a 4-inch diameter). Because the deposited Au layer badly adhered to the SiO2 surface, the Au/SiO2 interface could be exfoliated without significant mechanical deformation.The first approach utilizes a traditional TS process, as shown in The surface roughness of the fabricated films was evaluated using AFM. 2 template to the packaging substrate, as illustrated in 2 interface was mechanically exfoliated.Another approach is to transfer the Au/Ti/Au getter layer fabricated on the SiO2 template was rough because the RMS roughness was 3.48 ± 0.09 nm owing to grain growth during the deposition step. Meanwhile, the Au/Ti layer was atomically thin; the RMS roughness was 0.548 ± 0.01 nm. The surface of the transferred Au/Ti/Au getter layer is shown in AFM images of the Au/Ti/Au getter film before and after the transfer process are shown in 2/Si substrate with a 20/5-nm-thick Au/Ti bonding layer at room temperature, as illustrated in −2 Pa.To demonstrate the packaging process using the transferred Au/Ti/Au getter layer, the surface was bonded with another SiOThe room-temperature bonding of the transferred getter layer was evaluated by SAM. The mechanical strength of the bonded substrates shown in The interface formed in the room-temperature bonding experiment was observed using SEM and TEM. The cross-sectional surfaces of the diced chips were smoothened using an ion polisher. Nanoscale observations were performed using TEM, as shown in In this study, the thickness of the Ti layer was adjusted to 100 nm, which is the same as that in a previous study on the gettering process . However2 template, and the Au/SiO2 interface was mechanically exfoliated. Although this process reduced the surface roughness to 0.98 ± 0.07 nm, this value is undesirable for vacuum packaging. In the second approach, a 20/100/20-nm thick Au/Ti/Au getter layer was formed on the SiO2 template. This layer was transferred to the packaging substrate with an Au/Ti bonding layer, which was bonded to the Au/Ti bonding layer, and the smooth Au/SiO2 interface was exfoliated. The surface roughness of the transferred getter film was significantly reduced because the RMS surface roughness was 0.538 ± 0.12 nm. The results of the bonding experiments indicated that the transferred Au/Ti/Au getter layer could form wafer-scale direct bonding at room temperature; observations using electron microscopes suggests that ~10-nm-diameter voids were present at the bonding interface but not connected with others. Thus, it is expected that hermetic sealing without a leak path can be fabricated by the direct bonding of the transferred getter layers. We believe that this approach would allow for a simple packaging process for microsystems.In this study, an Au/Ti-based getter film was directly bonded to another substrate at room temperature for a simplified vacuum packaging process. Because hermetic sealing requires the bonding of atomically smooth Au surfaces, the fabrication processes of the smooth getter film were designed based on a TS technique. In the first approach, an Au/Ti getter layer with a thickness of 6/100 nm was deposited on a packaging substrate. The RMS roughness of the Au/Ti film was initially 2.00 ± 0.19 nm. This was bonded to a 20-nm-thick Au layer on a smooth SiO


Candida auris represents an emerging pathogen that results in nosocomial infections and is considered a serious global health problem. The aim of this work was to evaluate the in vitro antifungal efficacy of Cinnamomum cassia essential oil (CC-EO) pure or formulated in polycaprolactone (PCL) nanoparticles against ten clinical strains of C. auris. Methods: nanoparticles of PCL were produced using CC-EO (nano-CC-EO) and cinnamaldehyde (CIN) through the nanoprecipitation method. The chemical profile of both CC-EO and nano-CC-EO was evaluated using SPME sampling followed by GC-MS analysis. Micro-broth dilution tests were performed to evaluate both fungistatic and fungicidal effectiveness of CC-EO and CIN, pure and nano-formulated. Furthermore, checkerboard tests to evaluate the synergistic action of CC-EO or nano-CC-EO with micafungin or fluconazole were conducted. Finally, the biofilm disrupting activity of both formulations was evaluated. Results: GC-MS analysis shows a different composition between CC-EO and nano-CC-EO. Moreover, the microbiological analyses do not show any variation in antifungal effectiveness either towards the planktonic form (MICCC-EO = 0.01 ± 0.01 and MICnano-CC-EO = 0.02 ± 0.01) or the biofilm form. No synergistic activity with the antifungal drugs tested was found. Conclusions: both CC-EO and nano-CC-EO show the same antimicrobial effectiveness and are potential assets in the fight against C. auris.Background:  Candida auris has rapidly become a multidrug-resistant fungus of interest to global public health [First isolated in 2009, c health . SeveralC. auris to various healthcare facilities in Liguria and interregional spread to Emilia-Romagna, in order to contain any outbreaks and to deal with any national and international alert, the Ministry of Health identified the Complex Operating Unit of Microbiology of Policlinico Gemelli IRCCS in Rome (the laboratory of the microbiologists authors of this study) as a National Reference Laboratory for the characterization of C. auris isolates and the collection of clinical strains [In May 2018, the Italian Ministry of Health promptly issued the ECDC’s Rapid Risk Assessment and then subsequent recommendations aimed at raising awareness of this pathogen to increase surveillance and improve case management were issued ,4,5. In  strains .Resistance to antifungals, normally used as drugs or for surface treatment, is the basis of the high virulence and mortality. Most of the isolated strains are resistant to at least one class of antifungals, such as azoles, commonly used in therapy, and many of these isolates show multiple resistance, sometimes to all available drugs .This microorganism essentially affects immunocompromised people in nosocomial healthcare , and recC. auris is a challenge where laboratories, physicians, public health agencies and scientific research are needed; the former to identify and treat infections and prevent transmission, the latter to identify valid alternatives to current treatments capable of overcoming the resistance of the fungus.Therefore, appropriate control/containment measures using suitable room disinfection protocols are essential to reduce the spread. It has been demonstrated that chlorine-based products are the most effective for the disinfection of environments, while few and conflicting data are published regarding the efficacy of antiseptics  for decolonization of patients and hygiene of the hands of the healthcare personnel . TherefoC. auris. Drug reuse, combinations of new or old drugs, vaccine development, research of new synthetic  or natural  compounds [There are many lines of research aimed at identifying new resources in the fight against antibiotic resistance of ompounds ,11 are sC. auris and its biofilm [Cinamomum cassia EO (CC-EO) is one of the most powerful with its bioactive compound cinnamaldehyde [C. cassia Presl is a tropical aromatic evergreen tree of the Lauraceae family, commonly used in traditional Chinese medicine. It is also a traditional spice, widely used around the world. From its bark, it is possible to obtain an EO characterized by 70–90% cinnamaldehyde which defines its interesting anti-bacterial and anti-fungal activities [The essential oils (EOs) produced by the distillation of aromatic plants are, among the natural substances, the products with the highest antimicrobial potential ,13. Seve biofilm ,15,16,17aldehyde ,19. C. ctivities .Recent findings again prompt that EOs can function in a synergistic combination with antifungals ,22. RegaAmong the different polymeric materials, polycaprolactone (PCL) possesses excellent physicochemical characteristics for controlled release due to its high permeability to a wide range of drugs, a peculiar non-toxicity and a low degradation rate. Its good solubility in many solvents and its exceptional compatibility with various mixtures, including EOs ,29, has C. cassia EO and its nano formulation in PCL particles against clinical strains of C. auris by evaluating fungistatic and fungicidal activity.The aim of this article is to evaluate and compare the antifungal efficacy of The properties of nanoparticles, such as better selectivity and the ability to cross tissue barriers, are leading to the development of new techniques in the pharmacological field, for the development of innovative therapies. In the biomedical sector, one of the main applications concerns the formulation of new systems for the administration of drugs through nanoparticles in drug delivery systems. In this regard, monodisperse nanoparticles find interesting applications in nanomedicine . The intTrans-cinnamaldehyde was the main compound of the phytocomplex (85.5%) followed by O-methoxycinnamaldehyde (8.6%) and other minor molecules whose percentage values ranged from 0.1% to 0.9%.The chemical composition of CC-EO obtained by direct injection was characterized by 29 components listed in Headspace sampling followed by GC-MS analysis was carried out to evaluate the release of the volatile components from the nano-CC-EO in comparison with the free CC-EO one. The obtained results listed in CC-EO = 1 ± 0.00 and MICnano-CC-EO = 0.43 ± 0.12) and MIC values of fluconazole were detectable (MICFluconazole = 19.75 ± 20.50) even if they remained in the non-therapeutic range. In no case, a synergistic behavior was found.C. auris biofilm. Tested conditions were characterized by Fluconazole and Micafungin at a concentration of 256 μg/mL and 8 μg/mL, respectively, and by the free and nano formulated CC-EO at the MIC values (0.02% v/v) identified by micro broth-dilution tests. All treatments made with CC-EO showed a statistically significant activity (p < 0.001) when compared to untreated. No difference was observed comparing untreated control (CNT NT) with samples treated with the antifungals.C. auris. Towards C. auris strains, several EOs have been shown to have antifungal effectiveness when used alone  [Lavandula angustifolia, and Lippia sidoides) [Recently, several studies have been conducted in order to identify EOs potentially active against om bark) ,38 or inidoides) ,24.C. auris. As is known, CC-EO is a cheaper species than C. zeylanicum, which was selected in China to counter the East India Company English monopoly that made this spice very expensive in the 17th century [C. zeylanicum EO. In 2021, Xingdong Wu et al. identified some secondary metabolites  as the best markers to discriminate between the two cinnamon spices, which are otherwise difficult to distinguish even for most experts [C. cassia EO alone or encapsulated in PCL-based nanocapsules in order to identify if and in which form this EO can be used in the fight against C. auris. As shown in O-methoxycinnamaldehyde equal to 85.5% and 8.6%, respectively. These data are in line with ISO standards ISO 3216:1997 in which are specified the following standard ranges: 78.8–89.0% for cinnamaldehyde and 0.82–10.3% for O-methoxycinnamaldehyde [O-methoxycinnamaldehyde with a percentage of 0.7 compared to 1.0% in free CC-EO). Instead, the main chemical compound does not show significant variations . However, these differences do not affect the antifungal effectiveness of CC-EO. C. auris. Literature data indicate that CIN exerts its antifungal action through the inhibition of ATPases, the biosynthesis of the cell wall and the alteration of the structure and integrity of the membrane [C. auris has developed resistance, checkerboard tests were performed with both free and nano-formulated CC-EO. It was decided to use CC-EO as the phytocomplex is less toxic than the active compound alone as also reported by EMA monographs [To our knowledge, no studies have yet evaluated the activity of CC-EO against  century . EOs ext experts . Both EOaldehyde . PCL polaldehyde . PCL hasaldehyde  and theialdehyde ,42. As snographs . Due to CC-EO values indicate absence of synergies with both fluconazole and micafungin. Only the couple between CC-EO nano-formulated and micafungin showed, against resistant strains, a synergistic value of FICCC-EO that, however, is associated with a high variability (0.38) which makes the value borderline with the additivity. This behavior is different from that observed by our group testing the C. zeylanicum EO which, in agreement with literature data [p < 0.001) disrupting the biofilm if compared to antifungals that, on the contrary, fail to perturb it even if used at high concentrations. Therefore, our data show, for the first time, that the encapsulation process does not alter the properties of free CC-EO by upregulating or depressing its antimicrobial activity. This makes CC-EO encapsulated in PCL nanoparticles interesting because, as it is known in the literature, the use of polymeric and lipidic nanocapsules makes the administration of hydrophobic substances safer since, by increasing the circulating life, the solubility, the stability and the pharmacokinetic properties, reduces their systemic absorption and related side effects [The only two FICI values calculated for micafungin sensible strains indicate indifference between free or nano-formulated CC-EO when tested in combination with micafungin. Whereas, FICbility 0. which ma effects ,44,45. T effects .ε-caprolactone)  and sorbitan monostearate from Sigma–Aldrich ; Water Chromasolv Plus for HPLC solvent from Honeywell Riedel-de-Haën . All chemicals and solvents were of analytic or pharmaceutical grade.Polysorbate 80 was purchased from Fisher Chemical ; poly, and Sabouraud Dextrose Agar  were used to growth strains at 37 °C for 24 h.Ten Cinnamomum cassia essential oil  and cinnamaldehyde , its main component, were used for the study.The preparation of nano-capsules of CC-EO (nano-CC-EO) and those of cinnamaldehyde (nano-CIN) were performed by using the nanoprecipitation method as reported by Granata et al. . Brieflyv/v) with pure water or with pre-filtered (0.45 µm) 10 mM NaCl aqueous solution to perform DLS and ELS experiments, respectively. Data were analyzed using Zetasizer Version 7.02 software. DLS experiments provided the mean diameter (z-average) and polydispersity index (PDI) of nano-CC-OE and nano-CIN, while ELS provided the zeta potential (ζ). The encapsulation efficiency (EE), that represents an important parameter of the encapsulation process, was calculated using the following Equation (1):tot − bioactivefree is the amount of bioactive (CC-EO or CIN) into nano-CC-EO or nano-CIN, respectively. The total contents of CC-EO or CIN in the nanosuspensions bioactivetot were determined as reported by Granata et al. [max 284 nm. The amount of CC-EO or CIN was derived from this value compared to the related calibration curves (R2 ≥ 0.9994), obtained by plotting the absorbance at 284 nm of five solutions containing different concentrations of CC-EO or CIN (from 1.4 to 6.7 µg/mL). The total content of CC-EO or CIN in the nanosuspensions were 6.0 or 5.9 mg/mL, respectively. The amount of bioactivefree in nanosuspension was obtained by ultrafiltration/centrifugation technique using a Heraeus Pico 21 centrifuge . Initially, 500 µL of each sample were centrifuged for 15 min at 14,000× g and the supernatant were transferred to Nanosep® Centrifugal Devices with Omega™ Membrane 30K  and centrifuged at 3500× g for 180 min. Finally, to determine the free quantity of CC-EO or CIN in nanosuspension, 10 µL of ultrafiltered supernatant were diluted with 3 mL of acetonitrile and subjected to UV-visible measurements as reported above. The loading capacity (LC) of bioactive, was calculated by the following Equation (2):Dynamic light scattering (DLS) and electrophoretic light scattering (ELS) experiments were performed at 25 °C on a Zetasizer Nano ZS-90 . The nano-CC-EO and nano-CIN suspensions were previously diluted , at 70 eV in scan mode in the range 35–400 m/z. Relative percentages for quantification of the components were calculated by electronic integration of the GC-FID peak areas using the normalization method without the use of an internal standard and any factor correction. The identification of the separated compounds was performed by comparing the mass spectra for each compound with that reported on the MS library search (Wiley and Nist 02). Furthermore, linear retention index (LRI) of each compound was calculated using a mixture of n-alkanes  injected directly into GC injector at the same operating conditions reported above and each calculated LRI has been compared with those reported in Nist Chemistry WebBook. All analyses were carried out in triplicate.The chemical volatile fraction of free and nano CC-EO and the liquid phase of free CC-EO, performed by direct injection, was characterized by using a Clarus 500 model Perkin Elmer  gas chromatograph coupled with a mass spectrometer and equipped with a FID (flame detector ionization). For the separation of compounds, a Varian Factor Four VF-1 capillary column was housed in the GC oven. Briefly, the oven GC temperature program was: isothermal at 60 °C for 5 min, then ramped to 220 °C at a rate of 6 °C min6 cfu/mL were added to 50 μL of broth in which a known concentration of CC-EO, CIN or their nano-formulations was previously suspended. Starting from each natural product or nano-formulations, serial dilutions ranging from 0.6% v/v (6 mL/L) to 0.001% v/v (0.01 mL/L) were performed. Next, 0.05% v/v of Tween 80 was utilized as emulsifier, and the plate was incubated for 24 h at 37 °C. Positive controls were included. Minimum inhibitory concentration (MIC) values were visually determined. The MIC was defined as the lowest concentration with total inhibition of growth compared with the growth of positive control. The minimum fungicidal concentration (MFC) was evaluated by seeding and incubating 5 μL of the content of each well on Sabouraud Dextrose agar plates at 37 °C for 24 h. The MFC was defined as the lowest concentration with the death of almost the 99.9% of the initial inoculum. All tests were performed in triplicate.According to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) international guidelines, broth microdilution susceptibility tests were performed in 96 well-plate to evaluate the antifungal activity of CC-EO, CIN, and nano formulations of both (nano-CC-EO and nano-CIN). Briefly, 50 μL of a cell suspension equal to 1 × 10v/v (2.5 mL/L) to 0.002% v/v (0.02 mL/L) for CC-EO, from 0.2% v/v (2 mL/L) and 0.002% v/v (0.02 mL/L) for nano-encapsulates, from 256 μg/mL and 0.015 μg/mL for fluconazole, and from 4 µg/mL and 0.003 µg/mL for micafungin. Tests were performed using 96-well microplates , and incubated for 24 h at 37 °C. At the end of the incubation time, the MIC values relative to the single compounds and their synergies were measured to calculate the Inhibiting Fractional Concentrations (FIC) values and the related Indices of Inhibiting Fractional Concentrations (FICI) in accordance with the EUCAST international guidelines (22). The interactions between the various compounds were evaluated with to the FIC or FICI values as follows: synergy (FIC/FICI ≤ 0.5), additive effect (0.5 < FIC/FICI ≤ 1), indifference (1 < FIC/FICI ≤ 2), and antagonism (FIC/FICI ≥ 2).The checkerboard titration method was used to study the interaction between free or nano-encapsulated CC-EO with fluconazole or micafungin. Specifically, the following concentration ranges were tested: from 0.25% C. auris (strain CA10) was diluted in RPMI broth  in order to have 1 × 107 cfu/mL. The strain was grown overnight in 96-well plates , at 37 °C for 4 days. After the incubation period, the biofilm was treated for a further 24 h with or without CC-EO. Subsequently, biofilm was washed three times with PBS and cells fixated in acetone for 10 min. Each treatment was eightfold. The following treatments were studied: 256 μg/mL of Fluconazole, 8 μg/mL of Micafungin, and 0.02% v/v of CC-EO free and nano-encapsulated. The CNT NT was included. Crystal violet staining  was used to stain the resultant biofilm for 30 min. Finally, PBS was used to wash the biofilm, ethanol (100 μL/well) was added to completely dissolve the crystal violet, and the absorbance at 560 nm was detected.To evaluate the ability of CC-EO to disaggregate the biofilm, a suspension of 0.5 Mc Farland of a representative clinical high biofilm producer strain of p < 0.05) multiple comparison test was used to evaluate the effectiveness of nano-CC-EO vs. CNT NT on the biofilm demolition assay.Normal distribution data were analyzed using mean and standard deviation parameters. The GraphPad Prism v.8 software  was used to perform statistical analysis. An ordinary Dunnett’s (C. zeylanicum. Furthermore, in the extraction phase, the first has a higher yield than the last one and from both EOs with a superimposable chemical profile are extracted. Our data demonstrate that, against C. auris, CC-EO has interesting fungicidal activity both when used in free form and encapsulated in PCL particles. In fact, the encapsulation process does not alter the antimicrobial effectiveness of the EO and can contribute to a better delivery and lower toxicity of this EO consisting of cinnamaldehyde as the active compounds. To the best of our knowledge, this study demonstrates for the first time the potential efficacy of CC-EO in the fight against C. auris both when used as it is and encapsulated in PLC particles.CC-EO is an essential oil extracted from a cheaper species of the more valuable species of